Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling GATA2 + GATA3 with ab182747 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

Confocal image showing nuclear and weakly cytoplasmic staining on HeLa cells.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

The negative controls are as follows:-
-ve control 1: ab182747 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

Immunohistochemical analysis of paraffin-embedded Human prostate cancer tissue labeling GATA2 + GATA3 with ab182747 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on cancer cells of Human prostate cancer is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized K562 (Human chronic myelogenous leukemia cells from bone marrow) cells labeling GATA2 + GATA3 with ab182747 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

Confocal image showing nuclear and weakly cytoplasmic staining on K562 cells.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

The negative controls are as follows:-
-ve control 1: ab182747 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

Immunohistochemical analysis of paraffin-embedded Human neuroblastoma tissue labeling GATA2 + GATA3 with ab182747 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on cancer cells of Human neuroblastoma is observed.

Counter stained with Hematoxylin. 

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling GATA2 + GATA3 with ab182747 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on epithelial cells of mouse renal distal tubule is observed.

Counter stained with Hematoxylin. 

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Flow Cytometry analysis of K562 (human chronic myelogenous leukemia) cells labeling GATA2 + GATA3 with purified ab182747 at 1/150 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488)(1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labeling GATA2 + GATA3 with ab182747 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on rat epithelial cells of renal distal tubule is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Anti-GATA2 + GATA3 antibody [EPR17874] (ab182747) at 1/20000 dilution + Human GATA3 full length protein at 0.01 µg

Secondary
Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 51,48 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

Human GATA3 full length protein (epitomics/abcam) contains aa1-443 with N-terminal His-Tag®.

All lanes : Anti-GATA2 + GATA3 antibody [EPR17874] (ab182747) at 1/50000 dilution

Lane 1 : Empty vector (vector control) transfected HEK-293 (Human epithelial cells from embryonic kidney) whole cell lysate
Lane 2 : GATA2 transfected HEK-293 whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 51,48 kDa
Observed band size: 77 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Human GATA2 over-expression lysates (epitomics/abcam) contains aa1-480 with GFP-Tag.

All lanes : Anti-GATA2 + GATA3 antibody [EPR17874] (ab182747) at 1/10000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate
Lane 3 : MOLT-4 (Human lymphoblastic leukemia cell line) whole cell lysate
Lane 4 : LNCaP (Human prostate cancer cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 51,48 kDa
Observed band size: 51 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-GATA2 + GATA3 antibody [EPR17874] (ab182747) at 1/1000 dilution

Lane 1 : Human uterus lysate
Lane 2 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 51,48 kDa
Observed band size: 51 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-GATA2 + GATA3 antibody [EPR17874] (ab182747) at 1/1000 dilution + PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 51,48 kDa
Observed band size: 51 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-GATA2 + GATA3 antibody [EPR17874] (ab182747) at 1/1000 dilution + NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 51,48 kDa
Observed band size: 51 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

GATA2 + GATA3 was immunoprecipitated from 1mg of K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate with ab182747 at 1/100 dilution.

Lane 1: K562 whole cell lysate 10ug (Input).

Lane 2: ab182747 IP in K562 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab182747 in K562 whole cell lysate.

Western blot was performed from the immunoprecipitate using ab182747 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) was used as secondary antibody at 1/1500.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 30 seconds.